Process for separating micro-organisms from a fermentation wort

ABSTRACT

Yeast fermentation worts containing hydrocarbon, water, and inorganic salts, are resolved by contacting the worts with a mixed solvent system containing a dialkyl-ketone having at least four carbon atoms, e.g., methyl-ethyl-ketone, and another solvent selected from acetone, ethanol, n-propanol, iso-propanol and tert-butanol. As a result, three phases are formed, one of which is a heavy aqueous phase containing purified yeast which is then separated from the two other phases.

United States Patent [72] Inventors Claude Gatellier Boulogne; GeorgesGlikmans, Meudon la Foret, both of France [211 App]. No. 821,985

[22] Filed May 5, 1969 [45] Patented Dec. 7, 1971 [73] Assignee InstitutFrancais du Petrole, des

Carburants Et Lubriliant (Hts de Seine), France [32] Priority May 10,1968 [33] France [52] US. Cl 195/82,

Primary Examiner-A. Louis Monacell Assistant Examiner-Seymour RandAttorney-Milieu, Raptes & White ABSTRACT: Yeast fermentation wortscontaining hydrocarbon, water, and inorganic salts, are resolved bycontacting the worts with a mixed solvent system containing adialkyl-ketone having at least four carbon atoms, e.g.,methylethyl-ketone, and another solvent selected from acetone, ethanol,npropanol, iso-propanol and tert-butanol. As a result, three phases areformed, one of which is a heavy aqueous phase containing purified yeastwhich is then separated from the two t e phase -r PROCESS FOR SEPARATINGMICRO ORGANISMS FROM A FERMENTATION WORT This invention relates to a newprocess for separating microorganisms and more particularly yeasts froma fermentation wort; this process provides for recovery of themicro-organisms, particularly those produced in an aerated culture inmedia containing, in addition to the micro-organisms, one or morehydrocarbons, for example a hydrocarbon cut, and a nutritive aqueousmedium comprising inorganic salts as well as organic substances, forexample vitamins, which are necessary to the growth of micro-organisms.

It is well known that the culture medium recovered from a fermentationvessel, i.e., that vessel in which the culture takes place, consists ofa more or less thick emulsion which contains the micro-organisms, forexample yeasts, unconverted hydrocarbons (in the form of a freehydrocarbon phase or only of the portion adsorbed by the walls of thecells) and a substantial amount of aqueous inorganic medium. Aconventional centrifugation is usually unable to completely separate theyeasts from a substantial amount of hydrocarbons.

In order to purify the yeasts which thus have been obtained bycentrifugation, several treatments have been proposed, such as aplurality of washings with water containing a surface active agent,usually followed with a washing with pure water, each washing beingfollowed with a centrifugation.

Even after repeated washings, the yeasts remain insufficiently purified.

Another treatment consists of contacting the so-purified andcentrifugated yeasts with such solvents as saturated light hydrocarbons,for example hexane, aromatic hydrocarbons, for example benzene, ethers,alcohols, for example isopropanol or ethanol, or mixtures of the same.

Although these repeated washings and centrifugations are costly and timeconsuming, the purification of the yeasts is not completelysatisfactory.

According to this invention, an efficient separation and purification ofthe yeasts may be obtained by contacting the starting emulsion(comprising micro-organisms, water and hydrocarbons) with:

a. at least one dialkyl-ketone having at least four carbon atoms permolecule, for example methyl-ethyl-ketone, diethyl-ketone,methyl-isobutyl-ketone or propyl-ethylketone, and

b. at least one solvent selected from acetone, ethanol,

propanol, iso-propanol and tert-butanol.

Any type of contacting vessel may be used, provided an efficientstirring is obtained. Then the stirring is discontinued and 3 phasesappear: a heavy aqueous phase containing the microorganisms, for exampleyeasts, an intermediate phase containing water and the solvent of group(b), and a lighter phase which is rich in solvent of group (a) andresidual hydrocarbons.

The effluent from the fermentation vessel is preferably introduced atfirst into a quiet zone in order to obtain a first separation in twophases, one consisting of the so-called emulsion containing the residualhydrocarbons, the yeasts and a portion of the aqueous inorganic medium,the'other mainly consisting of the aqueous inorganic medium.

If a large amount of hydrocarbons remains unconverted at the end of theculture (for example when as substrate is used a gas oil containing aminor amount of straight-chain paraffins), three phases may appear: ahydrocarbon phase, an aqueous phase and the emulsion phase containingwater and a large amount of residual hydrocarbons.

Although the aqueous phase may be recycled to the fermentation vessel,the emulsion is collected in order to be subjected to the treatment ofthis invention.

It must be pointed out that this separation in three phases is obtainedonly with the above mixture of solvents. Thus when there is used forexample a mixture containing no solvent of the group (a), for instance amixture of acetone and isopropanol, the formation of three separatephases does not occur.

The temperature at which the mixture of solvents is contacted with theemulsion is preferably between 20 and 45 C., and more preferably atabout 30 C. The pH of the emulsion is preferably adjusted at a valuebetween 2 and 8 by any appropriate means.

In this process the yeasts may be separated from the fermentation wortwithout use of centrifugations whereby the cost of the process issubstantially reduced. At the same time the micro-organisms are washedby the solvents so as to make them free or substantially free fromhydrocarbons and organic byproducts resulting from the fennentation, forexample fatty acids, fatty alcohols or esters.

It is thus easy to recover, for example by mere decantation andwithdrawal, the heavy phase of yeasts from the three phases obtained bythis process.

The two other phases may also be easily separated, and, after removal,for example by distillation, of the residual hydrocarbons of thelightest phase and water of the intermediary phase, the solvents ofgroups (a) and (b) may be easily reused in a new separation andpurification step.

This treatment may be carried out in several manners. For example, theemulsion may be contacted with the solvent of group (a) and then withthat of group (b), or otherwise the emulsion may be contacted with thepreformed mixture of (a)+(b).

If desired, the paste of yeasts obtained by decantation according tothis invention may be thereafter contacted with a solvent or a mixtureof solvents such as those used by the prior art.

The yeast concentrate will be preferably contacted with a mixture ofsolvents which is identical to that used for separating the yeasts fromthe fermentation medium.

It will be mentioned that the separation process of this invention maybe applied not only to the emulsions containing hydrocarbons and yeasts,but also to the emulsions containing other micro-organisms such as moldsor bacteria. Thus any disclosure relating to yeasts" may also be appliedto molds or bacteria.

It is also possible to add a small amount of additional solvent, forexample a chlorinated hydrocarbon, to the mixture ofsolvents (a) and(b).

The total amount of solvents (a) and (b) to be contacted with theemulsion from the yeasts cultivation zone, and more particularly theratio of the solvents (a) and (b) are dependent on the amounts ofaqueous and hydrocarbon phases of this emulsion. I

It is preferred to use a small excess of solvent of type (a) withrespect to the solvent of type (b); for example the ratio by weight ofthe solvent of type (a) to the solvent of type (b) will .be between 1and 8 and more particularly between 1.5 and 5,

although these values are not considered as limitative.

As a rule, there will be used from 1 to 10 and preferably from 2 to 5volumes of mixture of the solvents (a) and (h) per volume of emulsioncontaining the micro-organisms.

This process may be carried out batchwise or continuously. It is ofparticularly high interest when the micro-organisms and moreparticularly the yeasts have been cultivated on a hydrocarbon substratecontaining a relatively low amount of straight-chain parafiins, forexample 5 to 69 percent by weight.

It is well known that the growth of yeasts is dependent on the normalparafiins content of the feed charge.

This process will thus be advantageously used for treating an emulsionfrom a fermentation vessel which had been fed, for'example, with aparafiinic gasoil (containing about 10 percent of normal paraffins).

The conditions under which the micro-organisms may grow are well known.For growing yeasts, the temperature is preferably between 20 and 40 C.,the pH preferably between 3 and 5 and a good contact must be realizedbetween oxygen, the yeasts and the hydrocarbon and aqueous phases.

The aqueous medium usually contains nitrogenand phosphorus-containingcompounds, for example (NH PO growth factors (compounds of the vitamin Btype), oligo-elements and essential ions such as Kfand S0,". Theessential ions are dependent on the particular type of cultivatedmicro-organisms.

Examplesl to 4 are given for illustrating the process of this invention;examples 1A and 4A are given for comparison purposes but form no part ofthis invention.

EXAMPLE 1 Yeasts of the Candida lipolytica type are continuously grownin a fermentation vessel, in the presence of a carbon source consistingof a gasoil containing 1 1 percent of linear parafi'inic hydrocarbonsand in the additional presence of an aqueous nutritive phase, thecomposition of which is given hereafter.

Air is injected in a divided form from the bottom of the vessel. The pHis maintained at about 4 and the temperature at about 27 C.

The aqueous nutritive medium has the following composition:

di-ammonium phosphate 2.2 g. potassium chloride l.l g. magnesium sulfate0.9 g. tap water (containing oligo-elements) 320 g. yeast extract(growth factor) 0.03 g. distilled water L000 g.

The liquid effluent from the fennentation vessel is passed into adecantation vessel in which a first separation takes place between anaqueous phase, half of which is recycledto the fermentation vessel, aphase of unconverted gasoil and an emulsion or paste of yeastsconsisting essentially of water (with inorganic salts), in a proportionof about 90 percent by weight, and of 5 percent of yeasts and 5 percentof residual'hydrocarbons.

One volume of this paste is contacted with 2 volumes of a liquid mixtureconsisting of 4 parts by volume of methyl-ethylketone and one part byvolume of acetone (the ratio by weight is also about 4/1 Three phasesseparate: a lower aqueous phase of yeasts, an intermediary phaseessentially comprising acetone and water and a lighter phase mainlycomprising methyl-ethyl-ketone and residual hydrocarbons.

The aqueous phase is withdrawn; the yeasts contained therein are free ofresidual hydrocarbons and of byproductsof the fatty acid type. They maybe dried and stored for a longtime without turning rancid.

The two other phases are distilled, and the thus recovered acetone andmethyl-ethyl-ketone may be used anew.

EXAMPLE 1 A Example 1 is repeated, except that the paste of yeasts iscontacted with 2 volumes of a mixture comprising 1 part of acetone and 4parts of iso-propanol.

The total volume of the emulsion increases but no separation of phasesoccurs as in example 1.

It is necessary to centrifugate the emulsion. Further the soobtainedyeasts still contain 1.2 percent of residual hydrocarbons.

The same operation has been repeated, without improved result, bychanging the amounts of acetone and iso-propanol.

EXAMPLES 2 AND 3 Example I is repeated with the following changes:

There is used a mixture of methyl-isobutyl-ketone and ethanol (example2) and a mixture of methyl-ethyl-ketone and propanol (example 3).

All other conditions remaining unchanged, particularly the amounts ofsolvents, there is obtained a separation of the emulsion in three phasesfrom which yeasts substantially free from hydrocarbons and tgproductsmay be obtained.

AMPLE 4 Example I is repeated with the following change: the hydrocarbonfeed charge for the culture step has been obtained by dewaxing a lubeoil. This charge contains percent of straight-chain paraffins. Theaqueous nutritive medium remains unchanged.

By decantation of the effluent from the fermentation vessel, there isobtained an emulsion consisting of 90 percent by weight of water, 9.5percent of yeasts and 0.5 percent of residual hydrocarbons.

One volume of this emulsion is contacted with 5 volumes of a mixtureconsisting of 1.5 parts by weight of methyl-ethylketone per part byweight of acetone.

As in example 1, three phases appear. The yeasts are removed from theheavy phase; they are substantially free from residual hydrocarbons andbyproducts.

EXAMPLE 4 A Example 4 is repeated except that the mixture ofmethylethyl-ketone and acetone is replaced by the same volume of amixture of hexane with ethanol.

The ratio by weight of hexane to ethanol is l.5, corresponding to aratio by volume of hexane to ethanol of 65/35.

Under these conditions, no separation in three phases occurs, but onlyan increase of the total volume of the emulsion. As in example 1 A it isnecessary to centrifugate in order to recover yeasts containing 0.2percent of residual hydrocarbons.

What we claim as this invention is:

l. A process for separating and purifying micro-organisms and moreparticularly yeasts from a fermentation wort also containing at leastone hydrocarbon and water together with inorganic salts, comprisingcontacting this wort with (a) at least one dialkyl-ketone having atleast four carbon atoms per molecule and (b) at least one solventselected from the group consisting of acetone, ethanol, n-propanol,iso-propanol and tert-butanol, said contacting resulting in theformation of three phases, one of which is a heavy aqueous phasecontaining purified yeasts, and separating the latter from the two otherphases.

2. A process according to claim 1, wherein the wort is subjected to aprior decantation step so as to separate at least one aqueous phase fromat least one emulsion of yeasts, the latter being contacted with thesolvents (a) and (b).

3. A process according to claim I, wherein the wort is contacted with apreformed mixture of the solvents (a) and (b).

4. A process according to claim I, wherein the dialkylketone ismethyl-ethyl-ketone, methyl-isobutyl-ketone, diethyl-ketone orpropyl-ethyl-ketone.

5. A process according to claim 1, wherein from i to 10 volumes of themixture of solvents (a) and (b) are used per volume of wort.

6. A process according to claim 1, wherein the ratio by weight of thesolvent (a) to the solvent (b) is between i and 8 at the time ofcontact.

7. A process according to claim 5, wherein from 2 to 5 volumes of themixture of solvents (a) and (b) are used per volume of wort.

8. A process according to claim 6, wherein the ratio by weight of thesolvent (a) to the solvent (b) is between 1.5 and 5 at the time ofcontact.

9. A process according to claim I, wherein the solvent (a) ismethyl-ethyl-ketone and the solvent (b) is acetone.

10. A process according to claim 1, wherein the wort is simultaneouslycontacted with a chlorinated hydrocarbon.

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2. A process according to claim 1, wherein the wort is subjected to aprior decantation step so as to separate at least one aqueous phase fromat least one emulsion of yeasts, the latter being contacted with thesolvents (a) and (b).
 3. A process according to claim 1, wherein thewort is contacted with a preformed mixture of the solvents (a) and (b).4. A process according to claim 1, wherein the dialkyl-ketone ismethyl-ethyl-ketone, methyl-isobutyl-ketone, diethyl-ketone orpropyl-ethyl-ketone.
 5. A process according to claim 1, wherein from 1to 10 volumes of the mixture of solvents (a) and (b) are used per volumeof wort.
 6. A process according to claim 1, wherein the ratio by weightof the solvent (a) to the solvent (b) is between 1 and 8 at the time ofcontact.
 7. A process according to claim 5, wherein from 2 to 5 volumesof the mixture of solvents (a) and (b) are used per volume of wort.
 8. Aprocess according to claim 6, wherein the ratio By weight of the solvent(a) to the solvent (b) is between 1.5 and 5 at the time of contact.
 9. Aprocess according to claim 1, wherein the solvent (a) ismethyl-ethyl-ketone and the solvent (b) is acetone.
 10. A processaccording to claim 1, wherein the wort is simultaneously contacted witha chlorinated hydrocarbon.